Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency.
The first practical test for the determination of creatinine in urine or serum, known as the Jaffe method, involves the formation of the red-yellowish brown colored creatinine picrate by the bonding of picric acid and creatinine in an alkaline solution. A more recent method for creatinine determination is reported by Benedict and Behre in J. Biol. Chem., 113:515 (1936) which involves the reaction of 3,5-dinitrobenzoic acid DNBA with creatinine in an alkaline medium. Each of these reactions require a high pH, i.e. greater than about 11.5 and typically from about 12 to about 14, in order to deprotonate the creatinine in order for the system to operate properly. Strongly basic substances such as alkali and alkaline earth metal hydroxides are typically used to maintain a suitably high pH in these reagent systems.
The dried creatinine reagent system comprising an indicator which forms a colored reaction product at an elevated pH and an alkaline reagent to raise the pH to the desired level when the reagent is rehydrolyzed can be applied to an absorbant carrier such as filter paper or a porous film. Typically, the reagent system is applied to the carrier in the form of separate solutions, aqueous for the alkali and organic for the indicator, with evaporation of the solvent to leave the residual dry reagent dispersed in the carrier. This sort of system is disclosed in Japanese Patent Application No. 62-287261 which also discloses treating the strip with an alkali soluble carboxylic acid derivative polymer to inhibit degradation of the strip by the strong alkali.
There are available diagnostic devices for the determination of protein, particularly human serum albumin (HSA), in urine. The determination of HSA in urine has clinical significance for the detection of the early stages of nephropathy which is an abnormal state of the kidney. A high percentage of individuals suffering from insulin dependent diabetes mellitus (IDDM) and noninsulin dependent diabetes mellitus (NIDDM) eventually secrete HSA at levels above those of the upper end of the normal population. This stage of "microalbuminuria" becomes progressively worse and typically leads to nephropathy. Since the kidney damage at the stage of microalbuminuria can be controlled or reversed by administering appropriate therapy, it is well recognized that measuring microalbuminuria is part of the comprehensive care of IDDM and NIDDM.
Other urine bound proteins, e.g. IgG, alpha-1-microglobulin, Bence-Jones protein and N-acetyl-b-D-glucoseaminidase, are useful as markers to detect and differentiate prerenal, glomeruler and postrenal forms of microalbuminuria. Proteinuria, which is indicated if the protein concentration in urine is greater than 30 mg/L, is the common symptom for a variety of different kidney diseases. Accordingly, there is a need on the part of nephrologists, diabetologists and cardiologists for test methods that are sensitive, specific and quantitative for the determination of these proteins in urine.
In order to increase the sensitivity and specificity of urinary protein assays and minimize the problem of high urine flow rate resulting in urine dilution, protein/creatinine ratios are used in urine protein assay results to normalize the urine concentration. Typical protein analyses involve immunoassays such as radioimmunoassay, enzyme immunoassay, latex assisted immunoassay and immunoturbidimetric assay. Since the commonly used Jaffe and Benedict-Behre creatinine assays are run at a high pH, the common practice in clinical laboratories is to run the protein and creatinine assays separately and then combine the values obtained from these assays to generate the protein to creatinine ratio. Since patients with high urine flow rates tend to have artificially low protein values due to the urine's dilution and since creatinine is a good marker for dilution of urine, the use of the protein/creatinine ratio eliminates the problem of urine dilution and gives a more accurate reflection of the true protein excretion rate.
In U.S. Pat. 5,385,847 there is disclosed a device which permits the determination of protein and creatinine in a single urine sample in a reaction vessel in which there is carried out an immuno assay for the protein in the vessel's first reaction zone followed by the creatinine determination in a second reaction zone which contains a dried indicator for the determination of creatinine as well as the dried basic reagent which is necessary for raising the pH of the reaction medium when the reagents are rehydrated by introduction of the liquid sample to be tested into the second reaction zone.
Whether the dry creatinine reagent (indicator and alkaline material) be in the strip form or located in the device of the '847 patent, whose disclosure is incorporated herein by reference, there exists a storage problem due to the formation of carbonic acid from atmospheric moisture and carbon dioxide in the area of the dried alkali which, over time, can cause its neutralization thereby limiting or destroying its ability to induce the high pH necessary for the determination of creatinine in the test fluid. Typically, these devices have been packaged with a desiccant to reduce moisture in the system to thereby inhibit the formation of carbonic acid. Examples of such desiccants include molecular sieves, silica gel, sodium sulfate, magnesium chloride, lithium chloride and triethylene glycol. The use of a desiccant which serves merely as a drying agent has not proved entirely satisfactory which observation caused us to search for a more satisfactory class of desiccant.
Soda lime, a mixture of sodium hydroxide and calcium oxide, is described in Hackh's Chemical Dictionary, 4.sup.th Edition, as a general absorbant for acid gases. In Kirk-othmer, Encyclopedia of Chemical Technology, 3.sup.rd Edition, there is described on pages 674-678 of Volume 16 a sodium chlorate based generator for the production of oxygen. The generator comes equipped with a filter system which includes soda lime for the removal of byproduct CO.sup.2 from the oxygen stream.